Cell type specific gene transfers using retroviral vectors containing antibody-envelope fusion proteins and wild-type envelope fusion proteins

ABSTRACT

A method of infecting target cells with retroviral vector particles having target cell specificity. The retroviral vector particles have a chimeric envelope protein consisting of an antigen binding site of an antibody or another peptide, fused to the envelope protein the retroviral vector. The antigen binding site or other peptide disrupts the natural viral receptor binding site. The method includes producing the retroviral vector and contacting the vector with the target cell such that the vector is internalized by the cell.

[0001] This application is a continuation-in-part of pending applicationSer. No. 08/933,616 filed Aug. 28, 1997, which is continuation ofapplication Ser. No. 08/205,980, filed Mar. 4, 1994, now abandoned,which is a continuation-in-part of application Ser. No. 07/979,619,filed Nov. 20, 1992.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to retroviral vector particles havingtarget cell specificity. The retroviral vector particles comprise aretroviral vector having a chimeric envelope protein consisting of anantigen binding site of an antibody or another peptide fused to theenvelope protein of the retroviral vector. The antigen binding site orthe other peptide replaces or disrupts the natural viral receptorbinding site. The resulting chimeric envelope is referred to as the“targeting envelope”. This invention relates to retroviral vectors thatcontain not only the targeting envelope but also wild-type envelopeprotein. The presence of wild-type envelope in addition to the targetingenvelope acts as a helper molecule by supplying a fully functionalmembrane fusion domain, which may be impaired in targeting envelopes.This helper function enables and/or enhances infection of cells that donot contain a receptor for the wild-type envelope but do contain areceptor for the binding for the targeting molecule. This invention alsorelates to a method for preparing the retroviral particles and for usingthe retroviral vectors to introduce genes into vertebrate cells.

[0004] 2. Description of the Background

[0005] The disclosures referred to herein to illustrate the backgroundof the invention and to provide additional detail with respect to itspractice are incorporated herein by reference. For convenience, thedisclosures are referenced in the following text and respectivelygrouped in the appended bibliography.

[0006] Retroviral vectors are the most efficient tools to introducegenes into vertebrate cells. Clinical experiments have been conducted touse retrovirus vectors to cure a genetic disease in humans (adenosinedeaminase (ADA) deficiency). Besides correcting inborn errors ofmetabolism, gene therapy is also being tested in clinical trials to curecancer and various other diseases (Science 1992, Vol. 258, pp. 744-746).

[0007] Retroviral vectors are basically retroviral particles thatcontain a genome in which all viral protein coding sequences have beenreplaced with the gene(s) of interest. As a result, such viruses cannotfurther replicate after one round of infection. Retroviral vectorparticles are produced by helper cells (FIG. 1). Such helper cells arecell lines that contain plasmid constructs, which express all retroviralproteins necessary for replication. After transfection of the vectorgenome into such helper cells, the vector genome is encapsidated intovirus particles (due the presence of specific encapsidation sequences).Virus particles are released from the helper cell carrying a genomecontaining only the gene(s) of interest (FIG. 1). In the last decade,several retroviral vector systems derived from chicken or murineretroviruses, have been developed for the expression of various genes(for reviews see Temin, 1987; Gilboa, 1990).

[0008] Retroviral vectors have several limitations. Besides the limitedgenome size that can be encapsidated into viral particles, the mostlimiting factor for the application of retroviral vectors is therestricted host range of the vector particle. Some retroviruses can onlyinfect cells of one species (ecotropic retroviruses) or even only onecell-type of one species (e.g., HIV). Other retroviruses have a verybroad host range and can infect many different types of tissues of manydifferent species (amphotropic retroviruses).

[0009] The initial step of retroviral infection is the binding of theviral envelope (env) glycoprotein to specific cell membrane receptors,the nature of which is unknown for most retroviruses. However, theinteraction of the viral env protein with the cell surface receptor isvery specific and determines cell-type specificity of a particular virus(Weiss et al., 1985). The envelope protein of all known retroviruses ismade up of two associated peptides, (e.g., gp70and p20(E) in SNV). Thesepeptides are derived by proteolytic cleavage from the same precursor(gPR90env) encoded by the retroviral env gene. One peptide p20(E), alsotermed TM, anchors the protein in the membrane of the virus and, asshown with HIV, mediates the fusion of the virus and cell membranes. Thesecond peptide gp70, also termed SU, mediates the binding of the virusto its receptor and, therefore, determines the host range (Weiss et al.,1985; Varmus and Brown, 1989).

[0010] Data obtained with several retroviruses indicate that theretroviral envelope protein forms trimers or tetramers. The formation oftrimers appears to be mediated by the TM peptide (reviewed in Hunter, E.et al., 1990). Targeting envelopes retain TM in order to (i.) maintain amembrane fusion function and (ii) maintain oligomerization. However,since X-ray pictures are not available, it is unclear whether or to whatdegree the construction of targeting-molecules impaired the structure ofthe membrane fusion domain.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 is a diagram illustrating helper cells expressingretroviral proteins: (A) Helper cells are made by the transfection ofplasmids expressing all retroviral proteins necessary to form infectiousvirus particles; (B) After transfection of the retroviral vector, thevector RNA genome is encapsidated into core structures: (C) Helper cellsthat contain a plasmid express a modified envelope gene.

[0012]FIG. 2 is a diagram illustrating plasmids expressing mutantenvelope genes of spleen necrosis virus (SNV).

[0013]FIG. 3 shows the sequence of the single chain antibody gene (scFv)against the hapten DNP.

[0014]FIG. 4 is a diagram illustrating helper cells expressing targetingenvelopes plus wild-type envelopes. Such helper cells are made by thetransfection of plasmids expressing the corresponding proteins: (A) Ahelper cell expressing all retroviral proteins necessary to form (1)retroviral core proteins and (2) targeting envelope; (B) Helper cellsthat contain targeting plus wild-type envelope are made by transfectingplasmids expressing genes encoding such proteins. After transfection ofthe retroviral vector that has the gene of interest, the retroviralvector RNA genome is encapsidated into retroviral vector particlesdisplaying the envelope.

[0015]FIG. 5 is a diagram of a eucaryotic gene expression vectorconstruct. The gene expression vector was derived from a similar vectordescribed by Sheay, W. et al., 1993.

[0016]FIG. 6 is a diagram illustrating plasmids expressing spleennecrosis virus, SNV, core structure proteins, wild-type envelopeproteins, and various targeting envelope proteins.

[0017]FIG. 7 is a restriction map of pRSV-scFvN29-gamma expressing theanti-Her2neu single chain antibody gene including the authentichydrophobic leader sequence.

[0018]FIG. 8 shows the sequence of the anti-Her2neu single chainantibody.

[0019]FIG. 9 is a restriction map of pTC53. PCR products of single chainantibody genes can be cloned into the NaeI site and the resultingconstruct expresses a chimeric envelope, which is transported throughthe ER and displayed on the surface of SNV derived retroviral vectorparticles.

SUMMARY OF THE INVENTION

[0020] The present invention pertains to a retroviral vector particlehaving defined target cell specificity mediated by the nature of thetargeting envelope which can be a chimeric protein consisting of anantigen binding site of an antibody or another peptide that binds to aspecific cell surface structure (e.g., the receptor binding domain ofanother virus) fused to carboxy terminal parts of the retroviralenvelope protein. The targeting envelope mediates the first step ofretroviral infection, which is the binding of the virus to a specificcell-surface receptor. The present invention also pertains to retroviralparticles that contain a wild-type envelope in addition to the targetingenvelope. The presence of the wild-type envelope serves to act as ahelper molecule to improve or supplement a functional membrane fusiondomain. Using target cells that do not contain a receptor for thewild-type envelope (e.g., SNV is not infectious for human cells), thewild-type envelope is only involved in the second step of retroviralinfection, which is the efficient fusion of the viral and the cellularmembranes. The present invention also pertains to the construction ofretroviral vectors particles containing a wild-type envelope in additionto a targeting envelope which can compensate for the loss of infectivityobserved with retroviral particles that contain targeting envelopesalone.

[0021] In one embodiment, the present invention pertains to a retroviralvector particle having target cell specificity which comprises aretroviral vector having a targeting peptide fused to the envelopeprotein of the retroviral vector to form a targeting envelope, whereinthe targeting peptide replaces or disrupts the natural viral receptorbinding site and the targeting peptide is the antigen binding site of anantibody, the receptor binding peptide of another virus, or is a peptidethat specifically binds to a specific receptor of the target.

[0022] In another embodiment, the present invention pertains to a celltype specific method for introducing genes into vertebrate cells usingretroviral vectors which comprises administering to the cells aretroviral vector particle having target cell specificity whichcomprises a retroviral vector having a targeting peptide fused to theenvelope protein of the retroviral vector to form a targeting envelope,wherein the targeting peptide replaces or disrupts the natural viralreceptor binding site and the targeting peptide is the antigen bindingsite of an antibody, the receptor binding peptide of another virus, oris a peptide that specifically binds to a specific receptor of thetarget.

[0023] In yet another embodiment, the present invention pertains to amethod for preparing a retroviral vector particle having target cellspecificity which comprises a retroviral vector having a targetingpeptide fused to the envelope protein of the retroviral vector to form atargeting envelope, wherein the targeting peptide replaces or disruptsthe natural viral receptor binding site and the targeting peptide is theantigen binding site of an antibody, the receptor binding peptide ofanother virus, or is a peptide that specifically binds to a specificreceptor of the target, which comprises the steps of:

[0024] (a) providing a targeting peptide;

[0025] (b) replacing part of the envelope gene coding for the viralreceptor binding site with the targeting peptide to form a chimericenvelope gene;

[0026] (c) cloning the chimeric envelope gene in a eucaryotic geneexpression vector; and

[0027] (d) transfecting the chimeric envelope expression plasmid, aretroviral core protein expression plasmid, and a selectable marker geneexpression plasmids into eucaryotic cells.

DETAILED DESCRIPTION OF THE INVENTION Targeting Envelope

[0028] This invention relates to retroviral vector particles havingtarget cell specificity. The retroviral vector particles comprise aretroviral vector having a chimeric envelope protein consisting of anantigen binding site of an antibody or another peptide fused to theenvelope protein of the retroviral vector. The antigen binding site orthe other peptide replaces or disrupts the natural viral receptorbinding site. The resulting chimeric envelope is referred to as the“targeting envelope”. This invention relates to retroviral vectors thatcontain not only the targeting envelope but also wild-type envelopeprotein. The presence of wild-type envelope in addition to the targetingenvelope acts as a helper molecule by supplying a fully functionalmembrane fusion domain which may be impaired in targeting envelopes.This helper function enables and/or enhances infection of cells that donot contain a receptor for the wild-type envelope but do contain areceptor for the binding of the targeting molecule. This invention alsorelates to a method for preparing the retroviral particles and for usingthe retroviral vectors to introduce genes into vertebrate cells.

[0029] To alter the host range of a vector particle, retroviral vectorparticles may be constructed that contain modified envelope proteinsthat recognize only a cell surface structure (receptor) specific for thetarget cell of interest. Proteins known to recognize specific structuresof proteins are antibody molecules. Hence, to make a retroviral vectorparticle specific for a cell-type of interest, the viral receptorbinding peptide may be replaced with an antigen binding site of anantibody molecule. To test whether vector particles containing suchantigen binding sites are competent for infection, model systems weredeveloped using an antigen binding peptide of an antibody against thehapten dinitrophenol (DNP) fused to envelope gene of spleen necrosisvirus (SNV).

[0030] The use of the anti-hapten (anti-DNP) antibody has manyadvantages. (1) The interaction of this antigen with the antibody iswell characterized (Davies and Metzger, 1983). (2) The hapten is easilyavailable. (3) A large variety of cells (which cannot be infected withwild-type vector particles) can be conjugated with this hapten. DNPconjugated cells bind antibodies directed against this hapten. Thus, thehapten may mimic the (abundant) presence of a receptor for the chimericvector particle. (4) Anti-hapten antibodies are frequently internalizedby the cell. Thus, the construction of chimeric envelope proteins willdestroy the membrane fusion domain of TM. This property may compensatefor this loss of function. (5) An in vitro binding assay can be easilyestablished to test for virus particle formation and binding of suchviruses to DNP. (6)

Wild-Type Envelope

[0031] This invention relates to retroviral particles having a targetcell specificity. The retroviral vector particles comprise a retroviralvector having a targeting envelope which mediates the binding of theretroviral vector particle to a cell surface receptor of the targetcell. This binding is very specific and determines the host range andcell-type specificity. The particles also have a wild type envelope.Using target cells that do not contain a viable receptor for the wildtype envelope, the function of the wild-type envelope is only to supplya fully functional membrane fusion domain. This invention also relatesto the method for preparing the retroviral vector particles and a methodfor using the retroviral vectors to introduce genes into vertebratecells.

[0032] Retroviral vectors derived from spleen necrosis virus containingwild-type envelope alone cannot infect human or hamster cells. In theseinfectivity studies, retroviral particles harvested from DSN cells wereused (Dougherty, J. P. and Temin, H. M. 1989) to infect human HeLa andCol-1, as well as hamster CHTG (ret. 1) cells (Tables 1 and 2). DSNcells are standard retroviral packaging cells containing a plasmidexpressing the retroviral core proteins and another plasmid expressingwild-type envelope (Dougherty, J. P. and Temin, H. M. 1989).

[0033] To introduce genes into such cells using SNV retroviral vectorparticles, two different approaches were made using different targetingenvelopes in combination with and without additional wild-type envelope.

[0034] 1. Targeting of human cancer cells (HeLa and Col-1) with SNVretroviral vectors. The antigen binding site of an antibody directedagainst the hapten DNP was used. In the experiments described below, theantigen binding site used in the targeting envelope was derived from anantibody (termed B6.2, Bird, R. E. et al., 1988 and Colcher, D. et al.,1990) directed against a cell-surface protein expressed on various humancancers (e.g., HeLa and Col-1 cells, Bird, R. E. et al., 1988 andColcher, D. et al., 1990). The gene constructs (FIG. 6) for theexpression of the targeting envelope are similar to that describedabove. In particular, in two constructs (FIG. 6, pTC24 and pTC25), theantibody moiety was fused to the same position of the SNV envelope geneas the anti-DNP antibody described below (for more details, see below:Material and Methods). To test whether the addition of a fullyfunctional membrane fusion domain (provided by wild-type envelope) wouldincrease the efficiency of infection, helper cells expressing retroviralcore proteins, wild-type envelope, and the targeting envelope weredeveloped (FIG. 4). Virus was harvested from such helper cells andsubjected to infectivity studies.

[0035] 2. Targeting CHTG cells that express a receptor for ecotropicmurine leukemia virus. To test whether retroviral particles derived fromSNV displaying targeting molecules other than antigen binding sites ofan antibody are infectious, targeting envelopes were constructed thatcontained the receptor binding peptide of another virus (murine leukemiavirus) fused to the envelope of SNV. Infectivity of virus particlesdisplaying such targeting envelopes with and without wild-type envelopewas tested.

EXAMPLES Targeting Envelope MATERIALS AND METHODS Construction ofAntibody-Envelope Fusion Genes

[0036] The gene coding for the envelope protein of spleen necrosis virus(SNV) does not contain suitable restriction enzyme sites to enable theconstruction of antibody-envelope fusion genes. Thus, point mutationswere introduced (by site directed mutagenesis) in the SNV env gene atdifferent locations to create restriction enzyme recognition sites. Forthis purpose, the SNV env gene (HindIII-SacI fragment) was subclonedinto pSelect (a vector specifically designed for site directedmutagenesis). Restriction sites for enzymes that create blunt ends wereintroduced in such a way that the restriction enzymes cut between twocodons. Following consistently this strategy, all mutants can be used tocreate deletions, insertions, and fusions in any combination withoutaltering the reading frame. Further, restriction enzyme sites werenested between regions coding for hydrophobic and hydrophilic domains.It was hypothesized that the deletion of a certain domain(s) would notinterfere with the proper folding of the following domain. Thishypothesis is based on the finding that many proteins in evolution aroseby exon shuffling of functional domains.

[0037] Some mutant envelopes that have been made are shown in FIG. 2.pSNV-env-mC (FIG. 2a) contains a new restriction enzyme site locatedbetween a hydrophobic and a hydrophilic peptide domain. In this mutant,the change in the nucleotide sequence does not alter the amino acidsequence. Thus, pSNV-env-mC can be considered as a positive control.pSNV-env-mD contains a new restriction enzyme site within the cleavagesite of the envelope precursor. The introduction of the mutation alsoaltered the amino acid sequence destroying the common motive found inall cleavage sites of all retroviruses investigated. Thus, it wasexpected that the resulting envelope precursor would not be cleaved,and, therefore, would not to give rise to infectious virus particles.Mutated env genes were inserted into pHB3, a eucaryotic gene expressionvector (FIG. 2).

[0038] The genes coding for the heavy and the light chain of an antibodyagainst DNP have been kindly provided by Dr. Ogawa (Scripps Clinic, LaJolla, Calif.). The genes were sequenced and published (Riley et al.,1986). Using PCR technology as described (Whitlow and Filpula, 1990), asingle chain antibody gene was constructed including the signal peptideagainst DNP. The PCR product was cloned into the SmaI site ofpBluescript. DNA sequencing confirmed the successful combination of thetwo gene segments coding for the variable regions of the antigen bindingpeptide. The complete sequence of the anti-DNP scFv gene is given inFIG. 3. A SacII (located in the polylinker of pBluescript) to SmaI(located in the 3′ PCR primer) fragment was inserted into eucaryoticexpression vectors replacing amino terminal parts of the envelope geneas follows: in PTC4, the SacII (located upstream of the ATG codon of theenv gene) to SmaI fragment of env was replaced with the scFv gene' inpTC5 the SacII to the MscI fragment of env was replaced with the scFvgene (FIG. 2C and 2D, respectively). After cloning, theantibody-envelope junctions were sequenced to verify the maintenance ofthe correct reading frame of the chimeric gene.

Binding Assays

[0039] The in vitro binding assays were performed in the followingmanner. DNP was conjugated to BSA (DNP-BSA was used to raise the initialantibodies from which the scFv genes have been derived). DNP-BSA wascoupled to activated Sepharose following the protocol recommended by thesupplier (Sigma). An Elisa assay with an anti-DNP antibody (kindlyprovided by Dr. S. Pestka) confirmed the successful coupling reaction.100 ml of tissue culture supernatant medium was incubated with 50 ml ofDNP-BSA-Sepharose for 30 minutes at 37° C. After incubation, thesepharose particles were pelleted by centrifugation in a Qualitronminicentrifuge for 30 seconds. The pellets were rinsed once with PBS.The PBS was removed and reverse transcription assays were performed byadding the reaction to the sepharose pellet. The reverse transcriptionassay was done using standard procedures; incorporation of 32PdTTP intocDNA was determined by TCA precipitation as described (Schleif andWensink, 1981)

Test for Infectivity of Particles Containing Antibody-Envelope FusionProteins

[0040] The envelope expression plasmids shown in FIG. 2 were transfectedinto D17 cells (a dog osteosarcoma cell-line) in contransfection withpBR1 and pJD214HY (FIG. 2), plasmids expressing the retroviral coreproteins, and containing a retroviral vector for the expression of thehygromycin phosphotransferase gene, respectively (see also FIG. 1).Cells were selected for hygromycin resistance. After selection forhygromycin resistance, virus was harvested from confluent cell culturesand infectivity assays were performed (see below). Infected target cellswere selected for hygromycin resistance (D17 cells were incubated withmedium containing 60 mg/ml hygromycin, CHO cells with medium containing250 mg/ml hygromycin). Hygromycin resistant cell colonies indicateinfectious virus particles.

[0041] Infectivity assays were performed on D17 and CHO cells with andwithout conjugated DNP. DNP was conjugated to cells as follows: Cellswere incubated with 500 ml of a solution containing 1.4 mg/ml DNBS (2,4,-Dinitrobenzene-sulfonic acid, 2-hydrate, purchased from Kodak) insodium cocodylate buffer (0.25M) for 3 to 5 minutes at room temperature.The conjugation reaction was stopped by adding 5 ml of medium to thecells.

[0042] Infections of non-conjugated cells were performed in the presenceof 50 mM polybrene using standard protocols. In the case of DNPconjugated cells, infection was performed without polybrene.

WILD-TYPE ENVELOPE MATERIAL AND METHODS ScA Targeting Vectors

[0043] To construct a targeting envelope containing the antigen bindingsite of an antibody directed against a cell-surface protein expressed onseveral human tumor cells, the corresponding single chain antibody gene(termed B6.2, Bird, R. E. et al., 1988 and Colcher, D. et al., 1990)made for expression in E.coli was modified in the following way: PCRtechnology was used to amplify the B6.2 scA gene using the originalE.coli expression plasmid as template (Bird, R. E. et al., 1988 andColcher, D. et al., 1990). The primers used had the following sequence:

[0044] Primer A: 5′ GGAGCGCTGACGTCGTGATGACCCAGTC 3′

[0045] Primer B: 5′ CCTCGCGATCCACCGCCGGAGACTGTGAGAGTGGTGC 3′

[0046] The PCR amplification results in a fragment that does not containthe bacterial ompA signal sequence and the stop codons present in theoriginal B6.2 gene (Bird, R. E. et al., 1988 and Colcher, D. et al.,1990). The PCR products were cloned into the Sma1 site of thepBluescript vector (Strata gene) and sequenced to verify a correctreading frame. The plasmid was termed pTC9. The B6.2 gene was isolatedby digesting the pTC9 plasmid with Eco47III plus NruI. The correspondingrestriction enzyme recognition sites have been introduced with theprimers used for PCR amplification. The B6.2 gene (the Eco47III to NruIfragment was cloned into pTC13, a gene expression vector (FIG. 5). Thecorresponding vector (termed pTC23) contains the ER transport signalsequence of the SNV envelope protein fused to the B6.2 gene to enabletransport through the endoplasmatic reticulum. The cloning reconstitutedthe NruI site at the 3′ end of the B6.2 gene. Carboxy terminal parts ofthe SNV envelope gene were isolated and fused to the B.2 gene (NruIsite) to give plasmids pTC24, pTC25, and pTG26 (FIG. 6). Theseconstructs are very similar to plasmids pTC4 and pTC5 which contain theanti-DNP antibody. In plasmid pTC26, the antibody is fused to codon 168of the SNV envelope.

Chimeric SNV-MLV Targeting Envelope

[0047] Targeting envelopes containing the receptor binding peptide ofanother virus were made as follows: the gene segment of ecotropic murineleukemia virus (a HindIII-BalI fragment comprising almost the completeregion coding for the SU peptide, including its ER transport signalsequence, Ott, D., and Rein, A. 1992) was isolated and inserted into thevectors pSNV-env-mC and pSNV-env-mD (pSNV-env-mC and pSNV-env-mD wasdescribed in FIG. 2) replacing the amino terminal parts of the SNVenvelope gene. The resulting constructs are identical to plasmids pTC4and pTC5, respectively, except that the anti-DNP antibody peptide(anti-DNP scA) is replaced by the receptor binding peptide of ecoMLV(FIG. 6, pSNV-MLV-chiC and pSNV-MLV-chi-D, respectively).

Experimental System

[0048] Briefly, helper cells were made as described above bytransfecting plasmids expressing retroviral gag-pol proteins, theretroviral targeting envelope, and the wild-type envelope into D17 cellsin co-transfection with a selectable marker to obtain helper cell linescontaining targeting envelope only or helper cells containing bothtargeting and wild-type envelope. Infectivity assays were performed on avariety of different cell-lines which included D17 cells, CHTG-cellsexpressing the ecotropic murine leukemia virus receptor (Albritton, L.M. et al., 1989) and human HeLa and Col-1 cells. Infectivity wasdetermined with a retroviral vector expressing the bacterialbeta-galactosidase gene as described (Mikawa, T. et al.).

RESULTS Targeting Envelope

[0049] In vitro binding assay. The in vitro binding assays showed thatonly cells transfected with pSNV-env-mD produce viral vector particlesthat contain a chimeric envelope able to bind DNP (see also Table 1).

[0050] Infectivity studies. The results of the infectivity experimentsare summarized in Table 1. Vector particles containing wild-typeenvelope (pSNV-env-mC) infected D17 cells with an efficiency of about10⁵ (=100,000) colony forming units per ml of tissue culture supernatantmedium. Such virus particles also infected D17 cells conjugated withDNP. However, the efficiency of infection was three orders of magnitudeless than that of cells not conjugated with DNP. This drop in virustiter is mainly due to difficulties of selecting DNP conjugated cellswith the antibiotic. It appears that the conjugation reaction makescells very vulnerable to the drug and more than 90% of the cells diedtwo to three days after the conjugation action. Virus particles withwild-type envelope do not infect CHO cells.

[0051] The mutation of the cleavage site of the envelope precursorprotein (SNV-env-mD) completely abolished infectivity. Only one colonywas observed in D17 cells not conjugated with DNP. This findingcoincides with earlier reports that mutations in the envelope precursorcleavage site lead to non-infectious virus particles. Cells transfectedwith pTC4 (FIG. 2) did not produce vector particles that were able toinfect D17 or CHO cells at significant efficiencies. Cells transfectedwith pTC5 produced virus particles unable to infect D17 or CHO cells.However, such particles significantly infected cells conjugated withDNP.

WILD-TYPE ENVELOPE

[0052] First, the presence of wild-type envelope in particles displayingan antigen binding site against DNP was tested to determine whetherthere would be an increase in the efficiency of infection of cellsconjugated with DNP. It was found that DNP conjugated HeLa cells couldnot be infected with vector virus particles that contained the wild typeenvelope alone. However, DNP conjugated cells could be infected withanti-DNP displaying retroviral vectors at a very low efficiency. Thetiter measured was about 10 infectious units per ml of tissue culturesupernatant medium. Virus particles that contained wild-type envelope inaddition to the targeting anti-DNP envelope infected cells 10 to 30times more efficiently. This data indicate that the presence ofwild-type envelope can increase the efficiency of infection of targetingvectors. Two additional sets of experiments using other targetingmolecules were performed to corroborate this finding.

[0053] 1. Infectivity studies with virus particles containingantibody-envelope fusion proteins. D17 cells, HeLa cells and Col-1 cellswere infected with virus particles displaying an antigen binding site ofan antibody (B6.2, Bird, R. E. et al., 1988 and Colcher, D. et al.,1990) directed against a cell surface protein expressed on various humancarcinoma cells. Vector virus particles were harvested from a variety ofdifferent helper cell lines (Table 2). All virus particles were carryinga vector transducing the bacterial beta-galactosidase gene. Infectivitywas determined by staining the cells with X-gal as described (Mikawa, T.et al.). The number of blue cell colonies was determined two to threedays after infection. The following virus particles were tested forinfectivity: virus particles that do not contain envelope (termed “noenv”), virus particles that contain wild-type envelope alone (termedwt-env—DSN), virus particles that contain targeting envelopes alonewhich are antibody-envelope fusion proteins (termed TC24, TC25, and TC26as described in FIG. 6), and particles that contain wild-type plustargeting envelopes (termed TC24+wt-env, TC25+wt-env, and TC26+wt-env).Particles that do not contain any envelope were found to be basicallynot infectious. Particles that contain wild-type envelope wereinfectious only on D17 cells which contain a viable receptor forwild-type SNV. The particles were not infectious on HeLa cells or Col-1cells. Particles that contained targeting envelopes only were infectiouson D17 and HeLa cells. The efficiency of infection on D17 cells was lessthan 5% of that of virus containing wild-type envelope. Such particleswere not infectious on Col-1 cells. The addition of wild-type envelopeincreased efficiency of infection 10 to 50 fold. Further, Col-1 cellsthat could not be infected with particles containing either envelopealone could be infected with particles containing both wild-type env andtargeting env. This data indicates that the wild-type envelope adds afunction improving or even completely enabling virus penetration (Table2). These data also show that the level of infectivity is dependent onthe position within the envelope gene at which the antibody is fused tothe envelope.

[0054] 2. Infectivity studies with virus particles containingSNV-MLV-fusion proteins. CHTG cells (described in Albritton, L. M. etal., 1989) expressing the receptor of ecotropic murine leukemia virus aswell as D17 cells were infected with virus harvested from cellsexpressing targeting envelopes (SNV-MLV-chi-C and SNVMLV-chic-D) aloneor from helper cells expressing targeting envelope plus wild-typeenvelope. Virus particles were carrying to hygromycin B resistance gene.Infected cells were selected from hygromycin resistance and the numberof hygromycin resistant cell colonies was determined. Retroviral vectorparticles containing the targeting envelope alone were not infectious.The particles became infectious after wild-type envelope was added tothe particles. Particles with wild-type envelope alone are notinfectious on CHTG cells (Table 3).

DISCUSSION Targeting Envelope

[0055] The data obtained with retroviral particles containingantibody-envelope fusion proteins showed that such particles arecompetent for infection. Surprisingly, TC4, a construct that containsthe scFv gene fused to env in the middle of SU did not give virusparticles capable of binding DNP. This may be due to an unstable SU-TMcomplex. This hypothesis is supported by the finding that such particlesfailed to bind to DNP-BSA-Sepharose. Low level infectivity of suchparticles on D17 cells may result from unspecific adsorption of virusparticles containing TM only. Unspecifically adsorbed virus particles(depleted of SU) may occasional penetrate the cell.

[0056] Cells transfected with pTC5 produce virus particles with chimericenvelopes without a functional retroviral membrane fusion domain. Thisassumption is based on the finding that virus particles containinguncleaved envelope precursor proteins (SNV-env-mD) are not infectious.However, it is known that some antibody molecules are internalized bycells after binding to cell surface by an unknown mechanism. The datashow that such an internalization mechanism might be sufficient to allowinternalization of the virus particle and the consequent establishmentof a successful infection.

Applications of Vector Particles With Antibody-Envelope Fusion Proteinsin Gene Therapy

[0057] In all applications of human gene therapy so far, the cells ofinterest were isolated from the patient, purified from other cell types,and infected in tissue culture with retroviral vector particles whichwere harvested from helper cells. After expansion of the treated cellsin tissue culture, they were re-injected into the patient. The infectionof cells has to be done in vitro, since the retroviral vector particlesused (derived from amphotropic murine retroviruses) have a broad hostrange. Thus, if injected directly into the blood stream of a patient,such virus particles would infect all kinds of tissue. Besides otherrisks, this treatment would be inefficient, since the chance that thegene will be delivered to its appropriate target cell is very low.

[0058] This clinical gene therapy protocol may be sufficient to obtaininsight into how efficient and how beneficiary gene therapy will be forthe patient. Indeed, the clinical data look very promising (Eglitis,personal communication). However, the current clinical protocol is verylaborious, time consuming, very costly, and, therefore, not suitable forgeneral clinical application. For general clinical application, it willbe necessary to inject the gene transfer vehicle directly into the bodyof the patient.

[0059] The development of a retroviral vector particle that only infectsone specific cell type, may allow the direct injection of the vectorinto the patient's blood stream. The development of vector particlescontaining antibody-envelope chimeras may be the first step towards thisgoal and may open a new area of possible applications of gene therapy invivo.

In Vivo Delivery of Therapeutic Genes with Retroviral Vectors thatDisplay Single Chain Antibodies

[0060] To test in vivo gene delivery with retroviral vector particlesthat display single chain antibodies on the viral surface, SCID micehave been used as a first model system. It is well known that retroviralvectors are rapidly inactivated by complement, if they are produced fromcells of a heterologous species. However, SCID mice do not have afunctional immune system.

[0061] Confirmation that SCID mouse serum does not inactivate SNVretroviral particles. In our experiments, retroviral vector particleswere harvested from DSH-cxl packaging lines (described in Martinez andDornburg, Virology, 208: 234-241, 1995; Chu and Dornburg, J. Virol. 69:2659-2663, 1995). These packaging cells transduce a retroviral vectorexpressing the bacterial β-galactosidase gene (lacZ gene). The plasmidpCXL (Mikawa T. D., et al., Exp. Cell Res., 195: 516-523, 1992) containsa SNV-derived retroviral vector expressing the lacZ gene. Aliquots of100 μl of the retroviral vector solution were prepared and incubatedwith 0.1, 1, 10, and 100 μl of SCID mouse serum for 10 minutes,respectively. In a second experiment, the same amounts ofheat-inactivated (60° C. for 30 minutes) SCID mouse serum were added to100 μl of vector stocks. Next, the mixtures were added to 2×10⁵ D17cells in infectivity assays. Two days after this infection the cellswere stained with x-gal and the number of blue colonies was determinedas described (Martinez and Dornburg, Virology, 208: 234-241, 1995; Chuand Dornburg, J. Virol 69: 2659-2663, 1995). No significant differencesin infectivity were observed between samples incubated with fresh orheat-inactivated serum. Thus, the conclusion can be made that SCID mouseserum does not contain antibodies and/or complement factors whichinactivate SNV retroviral particles.

[0062] Cell type specific gene delivery in vivo. In another experiment,hygromycin-resistant human COLO320DM cells, which express the cellsurface marker Her2neu, (ATCC accession No. CCL 220) orhygromycin-resistant A431 cells, which do not express Her2neu (ATCCaccession No. HB-9629), were injected into the peritoneum of mice. Thenext day, vector virus particles, which displayed anti-Her2neu singlechain antibodies (10⁶ infectious particles) were injected. Thepreparation of these virus particles is described below. The vectorvirus particles transduced a marker gene, the bacterialbeta-galactosidase gene, into infected cells. Thus, by staining cellsafter infection, gene transduced cells stain blue and, therefore, can beeasily detected.

[0063] Construction of packaging cell lines. The construction of stablepackaging cells, which produce retroviral vector particles displayingthe anti-Her2neu antibody followed the protocol described in detail inChu. T. -H. and Dornburg, R., J. Virol 71: 720-725, 1997. Briefly, usingthe dog D17 cell-derived cell line DSgp13-cxl which expresses theencapsidation-negative SNV gag-pol proteins and the packagableretroviral vector pCXL, cell lines were made which expressed thechimeric scA-env fusion proteins. For this, the scA-Env gene expressionvector pAJ7 (described below) was transfected into DSgp13-xcl cells inco-transfection with a plasmid expressing a selectable marker gene. Inthis experiment, we used the puromycin resistance gene, driven by theSNV promoter, contained on pRD118-puro, derived from pRD118 (Chu, T. H.et al., Bioteching, 18: 890-899, 1995). However, one skilled in the artwould be able to readily to select an appropriate selectable markergene. About 100 to 200 single antibiotic resistant cell colonies wereisolated for each transfection. Expression of the scA-Env protein wastested by ELISA assays and infectivity assays as soon as cells had beentransferred to 24 well plates. The reason for making helper cells thisway is that transfected plasmid DNAs often integrate into ratherinactive chromosomal sites are poorly transcribed.

[0064] Next, the SNV wild-type envelope gene expression vector pIM29(Martinez, I. and Dornburg, R., J. Virol. 69:4339-4346, 1995; andMartinez, I. and Dornburg, R. Virology 208:234-241, 1995) wastransfected into cell lines established from the single coloniesdescribed above. Again, the transfection was done by co-transfecting aplasmid expressing a selectable marker (e.g., the hygromycin Bphosphotransferase gene). 100 to 200 single cell colonies were isolatedand tested for infectivity on human target cells. Cell clones with thehighest infectivity were selected, re-cloned once or twice, and finallyused for in vivo experiments.

[0065] pAJ7 is a plasmid containing the anti-Her1neu scA fused to theSNV-Env-TM coding region. This plasmid is very similar to plasmid pTC25(FIG. 6). However, it was made in a slightly different way: First,referring to FIGS. 7 and 8, a DNA linker coding for the amino acidsala-gly-ala-ser-gly-ser was inserted at the carboxy terminal end of theanti-Her2neu scA gene (which contains the authentic hydrophobic leadersequence of the antibody-gene for transport through the ER) in plasmidN29-gamma to give plasmid pRD161 (not shown). A DNA fragment (SnaB1 toEco47III) isolated from pRD161 and which contained the completeanti-Her2neu scA (including the hydrophobic leader sequence) were clonedinto pTC53 (FIG. 9) digested with Sac2 (blunt ended) plus NaeI to giveplasmid pAJ7. DNA sequencing was performed after all cloning steps toverify the maintenance of the correct reading frame of genes coding forthe chimeric protein.

[0066] Plasmid pTC53 (FIG. 9) is a gene expression plasmid for the fastcloning and efficient expression of single chain antibody-envelopefusion proteins for display on retroviral particles. pTC53 has beenderived from pTC13 (Chu, T. -H. and R. Dornburg, 1995, J. Virol. 69:2659-2663; and Chu, T. -H., et al., 1995, Biotechniq. 18: 890-899). Itcontains the MLV-U3 promoter and the Adenovirus 2 tripartite leadersequence followed by a sequence coding for the hydrophobic leadersequence of the SNV envelope gene Next, the plasmid contains a uniqueNaeI site (which cuts between two codons) for cloning of single chainantibody genes (e.g. PCR products). Downstream of the NaeI site is a DNAlinker coding for the amino acid motif (gly₄-ser)₃ to enable flexibilityof the scA. This gly-ser linker is fused in frame to the completetransmembrane peptide (TM) coding region of the SNV envelope gene. Thepolyadenylation site downstream of the TM coding region has been derivedfrom simian virus 40 (SV40). The plasmid backbone is that of pUC19.

[0067] Concentration of vector virus stocks. Concentrated vector virusstocks were prepared as described by Chu and Dornburg, J. Virol.71:720-725, 1997. Briefly, to concentrate the virus particles byultrafiltration, 15 ml of supernatant was added to Amicon Centriprepfiltration devices containing 100 or 500 kD molecular weight cut-off(MWCO) membranes (Amicon, Beverly, Mass.). The 100 kD cut-off membranesare made of cellulose, the 500 kD membrane is made of polysulfone (DavidBrewster, Amicon, personal communication). The vector particle solutionwas centrifugated in a Sorvall RC5000B table top centrifuge withswing-out buckets at 3,000 rpm for 30 min at 20° C. The supernatantmedium was discarded and the solution was centrifugated at least onemore time for 10 minutes or until a final volume of 1 ml (or less, e.g.,0.7 ml) was obtained. The final concentrate was used immediately for theinfectivity experiments.

[0068] Confirmation of cell-tape specific gene delivery in-vivo. The dayafter vector virus injection, the mice were sacrificed. Human cells wererecovered by trypsin treatment of the peritoneum. The cells wereisolated and subjected to different antibiotic selection in tissueculture. Hygromycin selection enables the growth of human COLO320DMHer2neu+target cells or A431 Her2neu-non-target cells but not the mousecells. In both experiments, human cells started to grow as colonies in avast background of mouse cells. 4 days after selection, the cells werestained with x-gal. (Chu, T. -H. and R. Dornburg. 1997. J. Virol 71:720-725). We found that 2 to 3% of the COLO320DM target cells wereinfected in vivo. None of the A431 were infected. Furthermore, none ofthe mouse cells (mainly epithelial cells of the peritoneum) stained blueindicating that they were not infected. This data indicates that acell-type specific gene delivery can be obtained in vivo and that SCIDmice are suitable model systems to study in vivo gene delivery.

Wild-Type Envelope

[0069] Retroviral vector particles which display an antigen binding siteof an antibody can specifically infect cells that contain an antigenspecific for the antibody. However, the efficiency of the gene transfercan be low. We hypothesized that the fusion of the targeting peptide tothe envelope impaired the natural fusion function of the envelope whichis essential for efficient penetration of the virus. Thus, thehypothesis that the addition of a wild-type envelope may complement thisshortcoming was tested.

[0070] New retroviral vector particles containing two different types oftargeting envelopes were constructed. These targeting envelopes were (1)fusion proteins containing the antigen binding site of an antibody fusedto various carboxy terminal portions of the envelope protein of spleennecrosis virus, SNV; and (2) fusion proteins consisting of the receptorbinding domain of ecoMLV fused to various carboxy terminal portions ofthe SNV, similar to the antibody envelope constructs (FIG. 6).

[0071] Targeting envelopes alone are little or not infectious on cellsthat contain a receptor for the targeting envelope. The addition ofwild-type envelope to particles containing targeting envelopesdramatically increased or even completely enable infectivity on targetcells that could hardly or not at all infected with virus particlescontaining either envelope alone. This data show that the constructionof particles containing mixed envelopes dramatically improves theefficiency of gene transfer into specific target cells and, therefore,provides a valuable tool to introduce genes into specific target cells.

[0072] This method can be probably be improved by mutating the naturalreceptor binding domain of the wild-type envelope (e.g., by sitedirected mutagenesis). Using a wild-type envelope containing anon-functional receptor binding site in mixed envelope retroviral vectorparticles may enable to also target cells that contain a receptor forthe wild-type envelope without loosing target cell specificity. TABLE 1Infectivity of Retroviral Vector Particles On D17 and CHO Cells With andWithout DNP Conjugation* Envelope of Binding Virus titer (cfu/ml) CHO −CHO + virus particle to DNP D17 cells D17 + DNP DNP DNP SNV-env-mC nd10⁵ 10² 0 0 SNV-env-mD — 1  0 0 0 TC4 — 10   0 0 1 TC5 + 0 10² 0 10 # bymeasuring reverse transcriptase activity bound to DNP-BSA-Sepharoseparticles. nd: not determined; 0: no hygromycin resistant colonies weredetected. Virus titers are expressed as colony forming units (cfu) perml of tissue culture supernatant medium.

[0073] TABLE 2 Infectivity of Retroviral Vector particles Displaying theB6.2 Single Chain Antigen Binding Peptide Construct Titer (CFU/ml)gag-pol + D17 HeLa COL-1 no env 1 nd —* wt-env (dsn) 1,000 nd nd TC26 4010 nd TC24 20 5 nd TC25 45 20 nd TC26 + wt-env 1,100 90 45 TC24 + wt-env10,000 250 115 TC25 + wt-env 4,000 100 55 # after infection, cells werestained with X-gal. Blue cells indicate infected cells expressing thelacZgene. Virus titers are expressed as colony forming units per mltissue culture supernatant medium harvested from helper cells (cful/ml).

[0074] TABLE 3 Infectivity of Retroviral Vector Particles ContainingChimeric Envelope Proteins of MLV and SNV Enveleope of virus Virus titer(cfu/ml) particle cells D17 cells CHTG MLY-SNV-chiC nd nd MLV-SNV-chiDnd nd MLV-SNV-chiC + wt SNV 10⁶ 10³ MLV-SNV-chiD + wt SNV 10⁶ 10³ DSN10⁵ nd # wild type (wt) SNV. DSN cells are SNV based helper cellsexpressing gag-pol and SNV env from two different plasmid constructs.Virus titers are expressed as colony forming units (cfu) per ml oftissue culture supernatant medium. ND: no hygromycin resistant colonieswere detected using a total of 5 ml tissue culture medium.

[0075] The term “oligonucleotide” as used herein refers to primers,probes, oligomer fragments to be detected, oligomer controls, andunlabeled blocking oligomers. Oligonucleotide are molecules comprised oftwo or more deoxyribonucleotides or ribonucleotides. The term “primer”as used herein refers to an oligonucleotide, preferably anoligodeoxyribonucleotide, either naturally occurring such as a purifiedrestriction digest or synthetically produced, which is capable of actingas a point of initiation of synthesis when subjected to conditions inwhich synthesis of a primer extension product, which is complementary toa nucleic acid strand, is induced, i.e., in the presence of nucleotides,an agent for polymerization such as a DNA polymerase, and a suitabletemperature and pH. The primer must be sufficiently long to prime thesynthesis of extension products in the presence of the polymerizationagent. Methods for amplifying and detecting nucleic acid sequences bypolymerase chain reaction (PCR) are described in detail in U.S. Pat.Nos. 4,683,195, 4,683,202, and 4,965,288, which disclosures areincorporated herein by reference.

[0076]FIG. 1 is a diagram illustrating helper cells expressingretroviral proteins. A) Helper cells are made by the transfection ofplasmids expressing all retroviral proteins necessary to form infectiousvirus particles. One plasmid is designed to express all core/proteins(expression of gag and pol). The other plasmid is designed to expressthe envelope precursor/protein. Both plasmid constructs do not containretroviral cis/acting sequences for virus replication (e.g.encapsidation sequences, a primer building site etc.). Polyadenylationtakes place in non/retroviral polyadenylation recognition sequences. B)After transfection of the retroviral vector, the vector RNA genome isencapsidated into core structures. The helper cell is producingretroviral particles that only contain the vector genome with thegene(s) of interest. The vector contains all cis/acting sequences forreplication. Thus, in infected target cells, the vector genome isreverse transcribed and integrated into the genome. Due to the lack ofretroviral protein coding genes in the vector genome, no virus particlesare produced from infected target cells. C) Helper cells that contain aplasmid express a modified envelope gene. The helper cell is verysimilar to that shown above. However, chimeric envelope genes wereconstructed that contain the antigen binding domain of an antibody atthe amino terminus fused to the carboxy terminus of the envelope gene.Such particles may only bind to and infect target cells that contain anantigen structure which is recognized by the antibody moiety of thechimeric envelope protein.

[0077]FIG. 2 is a diagram illustrating plasmids expressing mutantenvelope genes of spleen necrosis virus (SNV). Genes are expressed fromthe Rous sarcoma virus promoter (RSV/pro) and polyadenylated within thepolyadenylation signal of herpes simplex virus thymidine kinase gene(TK/poly(A)). The polylinker of pBluescript was inserted between thepromoter and the polyadenylation sequence to allow the easy cloning ofgenes into this vector (plasmid sequences that abut the vector are notshown). a/b) point mutations were introduced into the env gene by sitedirected mutagenesis to create new restriction enzyme recognition sites(indicated by an *). All enzymes cut exactly between two codons creatingblunt ends for easy ligation without shifting the reading frame. c/d)chimeric envelope of containing an antigen binding peptide fused to thecarboxy terminus of env. e) pJD214Hy, a retroviral vector used in allstudies to test the transfer of genes by retroviral vector particles.

[0078]FIG. 3 shows the sequence of the single chain antibody gene (scFv)against the hapten DNP.

[0079]FIG. 4 illustrates retroviral packaging cells. A) A eucaryoticcell containing two different plasmids for the production of retroviralvector particle proteins. A retroviral vector transfected into suchcells and carrying the gene of interest is encapsidated by retroviralcore proteins. The envelope expression vectors expresses targetingenvelopes (e.g., an antigen binding peptide fused to the envelope).Virus particles are produced that infect a target cell only thatcontains a receptor specific for the antigen binding site. The helpershown under B) is similar to that shown above except that it alsocontains a gene expression vector coding for the wild-type envelope.Virus particles produced from such helper cells contain “mixed”envelopes which consist of the targeting envelope and the wild-typeenvelope. Formation of mixed oligomers is possible because both, thetargeting as well as the wild-type envelope contain a complete TMpeptide which mediates the formation of oligomers.

[0080]FIG. 5 illustrates a eucaryotic gene expression vector (pTC13) toobtain high level of gene products that contain an ER recognitionsequence. The vector shown has been derived from another gene expressionvector (termed pRD114) which is described in Sheay, W. et al., 1993. Thevector shown differs from pRD114 in that it contains a gene fragmentcoding for an ER recognition signal sequence to enable the transport ofproteins through the endoplasmatic reticulum. It contains tworecognition sites for the restriction enzymes NruI and StuI which cutbetween two codons downstream of the ER signal sequence coding region.DNA fragments coding for any peptide can be inserted into this sector.Translation of the inserted gene is terminated by using one of the threestop codons. MLV-U3-pro: promoter and enhancer of murine leukemia virus:Ad. V. leader: tripartite leader sequence of adenovirus; SV40 poly(A):polyadenylation signal sequence of simian virus 40;

[0081]FIG. 6 illustrates plasmid vectors expressing targeting envelopeproteins. The PCR product of the gene coding for the single chainantibody B6.2 (a Eco47III to NruI fragrment, see Material and Methods)was cloned into the NruI site of pTC13 (FIG. 5) to give plasmid pTC23.Carboxy terminal parts of the SNV envelope gene were isolated and clonedinto the NruI site downstream of the B6.2 antibody gene. The resultingtargeting antibody-envelope fusion gene of pTC24 and pTC25 are similarto pTC4 and pTC5. pTC24 and pTC25 retain exactly the same amount of SNVenvelope as pTC4 and pTC5, respectively. In pTC26 the antibody codinggenes abuts the envelope coding region at codon 167 of the envelopegene. Plasmids pSNV-MLV-chiC and pSNV-MLV-chiD are identical to pTC4 andpTC5, except that the antibody gene is replaced with a gene fragmentencoding for almost the complete SU peptide of MLV. In these clones theER transport signal sequence (L) is from MLV. MLV-pro: promoter andenhancer of murine leukemia virus; Avtl: tripartite leader sequence ofadenovirus; L: ER transport signal sequence, B6.2scFV: gene encoding thesingle chain antibody B6.2; poly(A) polyadeylation signal sequence ofSV40; SU surface peptide coding region of the SNV envelope; TM:transmembrane coding region of the SNV envelope; RSV: promoter andenhancer of Rous sarcoma virus.

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[0114] Throughout this application, various publications have beenreferenced. The disclosures in these publications are incorporatedherein by reference in order to more fully describe the state of theart.

[0115] The invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

1 7 1 28 DNA Artificial Sequence Primer A used for PCR amplification ofB6.2 scA gene 1 ggagcgctga cgtcgtgatg acccagtc 28 2 37 DNA ArtificialSequence Primer B used for PCR amplification of B6.2 scA gene 2cctcgcgatc caccgccgga gactgtgaga gtggtgc 37 3 800 DNA ArtificialSequence B6.2 gene encoding single chain antibody against hapten DNP 3actggaggct gatttttgaa gaaaggggtt gtagcctaaa agatgatggt gttaagtctt 60ctgtacctgt tgacagccct tccgggtatc ctgtcagagg tgcagcttca ggagtcagga 120cctagcctcg tgaaaccttc tctgactctg tccctcacct gttctgtcac tggcgactcc 180atcaccagtg gttactggaa ctggatccgg aaattcccag ggaataaact tgagtacatg 240gggtacataa gctacagtgg tagcacttac tacaatccat ctctcaaaag tcgaatctcc 300atcactcgag acacatccaa gaaccagtac tacctgcagt tgaattctgt gactactgag 360gacacagcca catattactg tgcaagatat ggtggtaact atgctatgga gtactggggt 420caaggaacct cagtcaccgt ctcctcagga ggtggcggta caggtggcgg aggtacaggc 480ggaggtggta gaattgtgat gacacagtct ccatcctccc tggctatgtc agtaggacag 540aaggtcacta tgagctgcaa gtccagtcag agccttttaa atagtagcaa tcaaaagaac 600tatttggcct ggtaccagca gaaaccagga cagtctccta aacttctggt atactttgca 660tccactaggg aatctggggt ccctgatcgc ttcataggca gtggatctgg gacagatttc 720actcttacca tcagcagtgt gcaggctgaa gacctggcag attacttctg tcagcaacat 780tatagcactc cgtggacgtt 800 4 220 DNA Artificial Sequence pTC13 eucaryoticgene expression vector 4 gagctccacc gcggtaaagg tcgctgggaa gaccccgtggatccaccact ctcgactcaa 60 gaaagctcct gacaaccaag aagaatggac tgtctcaccaacctccgatc cgctgagggt 120 aaagttgacc aggcgagcaa aatcctaatt ctccttgtggcttggtgggg gtttgggacc 180 actgccgaag tttcgcgaag gccttaagtg actaggtacc220 5 40 PRT Artificial Sequence ER recognition signal sequence 5 MetAsp Cys Leu Thr Asn Leu Arg Ser Ala Glu Gly Lys Val Asp Gln 1 5 10 15Ala Ser Lys Ile Leu Ile Leu Leu Val Ala Trp Trp Gly Phe Gly Thr 20 25 30Thr Ala Glu Val Ser Arg Arg Pro 35 40 6 1079 DNA Unknown DNA encodinganti-Her2neu single chain antibody 6 ggatctacgt accatggatt ttcaggtgcagattttcagc ttcctgctaa tcagtgcctc 60 agtcataatg tctagaggag atattgtgatgacccagtct ccaaaattca tgtccacatc 120 agtaggagac aggatcagcg tcacctgcaaggccagtcaa gatgtgggtc ctaatgtagc 180 ctggtatcaa cagaaaccag ggcaatctcctaaaccactg atttactcgg catcctacct 240 atataatgga gtccctgatc gcttcacaggcagtggatct gggacagatt tctctctcac 300 catcagcaat gtgcagtctg atgacttggcagagtatttc tgtcagcaat ataacaccta 360 tccgttcacg ttcggagggg gcaccaagctggaaatcaaa gggtcgactt ccggtagcgg 420 caaatcctct gaaggcaaag gtgaggtgcagctggaggag tctggtggag gattggtgca 480 gcctaaaggg tcattgaaac tctcatgtgcagcctctgga ttcaccttca atacctacgc 540 catgaactgg gtccgccagg ctccaggaaagggtttggaa tggattgttc gcataagaag 600 taaaagtaat aattatgcaa catattatgtcgattcagtg aaagacaggt tcaccatctc 660 cagagatgat tcacaaagca tgctctatctgcaaatgaac aacttgaaaa ctgaggacac 720 agccatgtat tactgtgtga cttcttactatgattacgac aaggtcctgt ttgcttactg 780 gggccaaggg accacggtca ccgtctcttcacccgatcct cagctctgct atatcctgga 840 tgccatcctg tttctgtatg gaattgtcctcaccctcctc tactgtcgac tgaagatcca 900 agtgcgaaag gcagctataa ccagctatgagaaatcagat ggtgtttaca cgggcctgag 960 caccaggaac caggagactt acgagactctgaagcatgag aaaccaccac agtagcttta 1020 gactcgagta gatccagaca tgataagatacattgatgag tttggacaaa ccacaacta 1079 7 332 PRT Unknown Anti-Her2neusingle chain antibody 7 Ala Asp Phe Gln Val Gln Ile Phe Ser Phe Leu LeuIle Ser Ala Ser 1 5 10 15 Val Ile Ala Ser Arg Gly Asp Ile Val Ala ThrGln Ser Pro Lys Phe 20 25 30 Ala Ser Thr Ser Val Gly Asp Arg Ile Ser ValThr Cys Lys Ala Ser 35 40 45 Asp Val Gly Pro Asn Val Ala Trp Tyr Gln GlnLys Pro Gly Gln Ser 50 55 60 Pro Lys Pro Leu Ile Tyr Ser Ala Ser Tyr LeuTyr Asn Gly Val Pro 65 70 75 80 Asp Arg Phe Thr Gly Ser Gly Ser Gly ThrAsp Phe Ser Leu Thr Ile 85 90 95 Ser Asn Val Gln Ser Asp Asp Leu Ala GluTyr Phe Cys Gln Gln Tyr 100 105 110 Asn Thr Tyr Pro Phe Thr Phe Gly GlyGly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Ser Thr Ser Gly Ser Gly LysSer Ser Glu Gly Lys Gly Glu Val 130 135 140 Gln Leu Glu Glu Ser Gly GlyGly Leu Val Gln Pro Lys Gly Ser Leu 145 150 155 160 Lys Leu Ser Cys AlaAla Ser Gly Phe Thr Phe Asn Thr Tyr Ala Ala 165 170 175 Asn Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Ile Val Arg 180 185 190 Ile Arg SerLys Ser Asn Asn Tyr Ala Thr Tyr Tyr Val Asp Ser Val 195 200 205 Lys AspArg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ala Leu Tyr 210 215 220 LeuGln Ala Asn Asn Leu Lys Thr Glu Asp Thr Ala Ala Tyr Tyr Cys 225 230 235240 Val Thr Ser Tyr Tyr Asp Tyr Asp Lys Val Leu Phe Ala Tyr Trp Gly 245250 255 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Asp Pro Gln Leu Cys Tyr260 265 270 Ile Leu Asp Ala Ile Leu Phe Leu Tyr Gly Ile Val Leu Thr LeuLeu 275 280 285 Tyr Cys Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile ThrSer Tyr 290 295 300 Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr ArgAsn Gln Glu 305 310 315 320 Thr Tyr Glu Thr Leu Lys His Glu Lys Pro ProGln 325 330

What is claimed is:
 1. A method of infecting a cell having a selectedantigen comprising: (a) producing a retroviral vector having an envelopeprotein wherein a viral receptor portion is at least partially definedby a single chain antibody and said viral receptor portion binds to saidselected antigen: and (b) contacting said retroviral vector with thecell such that said viral particle or a portion thereof is internalizedinto the cell; wherein the cell having the selected antigen is infectedby said retroviral vector.
 2. The method of claim 1 wherein saidretroviral vector is produced using a plasmid comprising a nucleotidesequence encoding a single molecule comprising a portion of an envelopeprotein and a portion of a single chain antibody.
 3. The method of claim2 wherein said nucleotide sequence encodes a portion of the envelopeprotein of Spleen Necrosis Virus.
 4. The method of claim 2 wherein theplasmid is that identified in FIG. 2d as pTC5.
 5. A method of infectinga cell having a selected antigen comprising: (a) producing a retroviralvector having a targeting peptide fused to the envelope protein of saidretroviral vector to form a targeting envelop; and (b) contacting saidretroviral vector with the cell such that said viral particle or aportion thereof is internalized into the cell; wherein the cell havingthe selected antigen is infected by said retroviral vector.
 6. Themethod according to claim 5 wherein said targeting peptide replaces ordisrupts the natural viral receptor binding site.
 7. The method of claim5 wherein said targeting peptide is the antigen binding site of anantibody.
 8. The method of claim 5 wherein said targeting peptide is thereceptor binding peptide of another virus.
 9. The method of claim 5wherein said targeting peptide is a peptide that specifically binds to aspecific receptor of the target.
 10. The method of claim 5 wherein saidretroviral vector particle is spleen necrosis virus.
 11. The methodaccording to claim 5 wherein said the targeting peptide is a singlechain antibody against the hapten dinotrophenol (anti-DNP-scFv).
 12. Themethod according to claim 5 wherein the targeting peptide is an antigenbinding site directed against a cell-surface protein of the target cell.13. The method according to claim 5 wherein the retroviral vectorcomprises a targeting envelope and a wild-type envelope.
 14. A cell typespecific method for introducing genes into vertebrate cells usingretroviral vectors which comprises administering to the cells aretroviral vector particle having target cell specificity whichcomprises a retroviral vector having an antigen binding site of anantibody fused to the envelope protein of the retroviral vector, whereinthe antigen binding site of the antibody replaces the natural viralreceptor binding site.
 15. The method according to claim 14 wherein theretroviral particle is spleen necrosis virus.
 16. The method accordingto claim 14 wherein the antibody is a single chain antibody against thehapten dinitrophenol.
 17. The method according to claim 14 wherein thetargeting peptide is an antigen binding site directed against acell-surface protein of the target cell.
 18. The method according toclaim 14 wherein the targeting peptide is the receptor binding peptideof another virus.
 19. The method according to claim 14 wherein theretroviral vector comprises a targeting envelope and a wild-typeenvelope.
 20. The method according to claim 19 wherein the wild typeenvelope is derived from spleen necrosis virus.